Long term potentiation (LTP) of synaptic transmission is a form of synaptic plasticity that is observed in all principal neurons in the hippocampus-a brain structure implicated in certain forms of long-term memory. LTP has been extensively studied at Schaffer collateral and perforant path synapses, while, for technical reasons, relatively less attention has been paid to mossy fiber synapses in the hippocampus. The mossy fiber synapse has a number of unusual features, including LTP that does not depend on the NMDA type of glutamate receptor, and the mechanisms for the induction and expression of LTP at this synapse are not well understood. In this renewal application we propose to utilize visual patch-clamp techniques, pre- and postsynaptic Ca2+ imaging, focal stimulation of mossy fibers, and whole-cell recordings from single, mossy fiber boutons, all in acute slices of rat hippocampus, to investigate a number of hypotheses derived from our previous studies of this synapse. The aims of this proposal are: 1) To test the hypothesis that Ca2+ is released from stores inside mossy fiber boutons during stimulus protocols that induce LTP; 2) To test the hypothesis that Ca2+ released from intracellular stores postsynaptically during certain stimulus protocols is required for LTP induction; 3) To test the hypothesis that activation of protein kinases A, C, CaMKII, and MAPK is required for LTP induction or expression; 4) To test the hypothesis that brief-train LTP is associative and requires concomitant activity of other pathways for induction, while long-train LTP does not require co-activity of other afferents; and 5) To test hypotheses for a role of mossy fiber activity in the induction of LTP at commissural/associational and perforant path synapses onto CA3 neurons. The so-called "detonator synapse" hypothesis will be tested as part of this aim. We will also test for associative and/or heterosynaptic plasticity between the other synaptic inputs to CA3 neurons and the mossy fibers. We believe that the results of these studies will add significantly to our understanding of the computational properties of the hippocampus and the role of the hippocampus in learning and memory and diseases of cognition.